Contractile force in muscle fibers is generated by cross-bridges that form between the myosin-containing and actin-containing filaments. During shortening, the two types of filaments slide relative to each other, and the cross-bridges cycle between "attached" and "detached" states. The structural changes associated with cross-bridge formation are measured by X-ray diffraction techniques, using radiation from both rotating anode generators and synchrotron sources. The diffraction patterns are recorded with both one-dimensional electronic, position-sensitive, X-ray detectors and with film. Two-dimensional, electronic detectors are being developed. Muscle preparations are made from rabbits and frogs, and they include single skinned fibers, small bundles of skinned fibers, and intact muscles. The detailed structure of cross-bridges that form under various conditions (rigor (ATP-free), low ionic strength relaxed, calcium-activated) will be studied by analysis of the corresponding X-ray diffraction patterns. Correlations will be sought between the X-ray data and the mechanical and biochemical properties of the cross-bridges. The possible involvement of the two-headedness of the myosin molecule in force generation will be considered. The geometry of the rigor cross-bridge will also be examined by measuring the length of muscle fibers in the rigor state as a function of the myofilament lattice spacing derived from X-ray data. Kinetic properties of the cross-bridges are studied by analyzing the transient and steady-state contraction kinetics, and the stiffness, of skinned fibers under controlled chemical conditions. The influence of motion on the study number of cross-bridges is also followed by X-ray diffraction. These data provide information regarding the molecular basis of the force-velocity relation that characterizes the physiological behavior of skeletal muscle. The overall goal of the project is to find out how the actomyosin system in muscle fibers produces force and motion, and how the mechanical responses are regulated.